Vitamin D has a direct immunomodulatory effect on CD8+ T cells of patients with early multiple sclerosis and healthy control subjects.

Little is known on a putative effect of vitamin D on CD8+ T cells. Yet, these cells are involved in the immmunopathogenesis of MS. We assessed the cytokine profile of EBV-specific CD8+ T cells of 10 early MS patients and 10 healthy control subjects with or without 1,25(OH)(2)D(3) and found that, with 1,25(OH)(2)D(3), these cells secreted less IFN-γ and TNF-α and more IL-5 and TGF-β. CD4+ T cell depletion or even culture with CD8+ T cells only did not abolish the immunomodulatory effect of 1,25(OH)(2)D(3) on CD8+ T cells, suggesting that 1,25(OH)(2)D(3) can act directly on CD8+ T cells.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

Lack of directed V alpha 14-J alpha 281 rearrangements in NK1+ T cells.

NK1.1+ T cells are an unusual subset of TCR alpha beta cells distinguished by their highly restricted V beta repertoire and predominant usage of an invariant V alpha 14-J alpha 281 chain. To assess whether a directed rearrangement mechanism could be responsible for this invariant alpha chain, we have analyzed V alpha 14 rearrangements by polymerase chain reaction and Southern blot in a panel of cloned T-T hybrids derived from thymic NK1.1+ T cells. As expected a high proportion (17/20) of the hybrids had rearranged V alpha 14 to J alpha 281. However, V alpha 14-J alpha 281 rearrangements always occurred on only one chromosome and were accompanied by other V alpha-J alpha rearrangements (not involving V alpha 14) on the homologous chromosome. These data argue that rigorous ligand selection rather than directed rearrangement is responsible for the high frequency of V alpha 14-J alpha 281 rearrangements in NK1.1+ T cells.

Selective lysis of activated cells in oncorna virus infection.

The authors devised a cytotoxic assay based on cytofluorometric analysis of target surface markers in order to compare lysis exerted in vitro by cytotoxic T lymphocytes (CTLs) on different cell subsets in the context of a single lymphoid target cell population. Using this assay, the authors evaluated when oncorna virus-infected lymphocytes become a suitable target for virus-specific T cell effectors. A lymphocyte population from Moloney-murine leukaemia virus (M-MuLV)-infected (carrier) mice, in which the proliferation of selective V beta T-cell receptor (TCR) families was induced in response to Mlsa encoded antigens, was utilized as a target. The authors observed that a virus-specific T cell clone exerted lytic activity preferentially against activated cell subsets. Moreover, virus-specific CTLs generated in mixed leucocyte tumour cell cultures (MLTC) were also able to impair the concomitant anti-Mlsa response of lymphocytes from M-MuLV carrier mice. It was found that the proliferative status of oncorna virus-infected target cells played an important role in limiting the in vitro efficacy of the immune response, and it is speculated that this phenomenon might represent an in vivo escape mechanism from immunosurveillance.

Vaccination in murine schistosomiasis with adult worm derived antigens - II. Protective and immune response in inbred mice

Previous work in our laboratory, mainly foccused the prospects of achieving resistance against Schistosoma mansoni infection with adult worm-derived antigens in the form of a soluble extract (SE). This extract obtained by incubation of living adult schistosomes in saline, contains a large number of distinct molecules and was actually shown to be a significantly protective in different outbred animals models such as Swiss mice and rabbits. It thus appeared worthwile to investigate the potencial protective activity of SE in different inbred strains of mice, known to be highly susceptible to the infection. Herein we present data showing that DBA/2 mice, once immunized with SE acquire significant levels of resistance to a S. mansoni cercarial challenge. In addition, preliminary studies on the immune system of immunized animals reveled that, injection of SE caused no general inbalance of B or T cell responses.

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.

Cutting edge: NKT cell development is selectively impaired in Fyn- deficient mice.

Most NK1.1+ T (NKT) cells express a biased TCRalphabeta repertoire that is positively selected by the monomorphic MHC class I-like molecule CD1d. The development of CD1d-dependent NKT cells is thymus dependent but, in contrast to conventional T cells, requires positive selection by cells of hemopoietic origin. Here, we show that the Src protein tyrosine kinase Fyn is required for development of CD1d-dependent NKT cells but not for the development of conventional T cells. In contrast, another Src kinase, Lck, is required for the development of both NKT and T cells. Impaired NKT cell development in Fyn-deficient mice cannot be rescued by transgenic expression of CD8, which is believed to increase the avidity of CD1d recognition by NKT cells. Taken together, our data reveal a selective and nonredundant role for Fyn in NKT cell development.

Unexpectedly late expression of intracellular CD3epsilon and TCR gammadelta proteins during adult thymus development.

During adult thymus development immature CD4(-)CD8(-) [double-negative (DN)] precursor cells pass through four phenotypically distinct stages defined by expression of CD44 and CD25: CD44(hi)CD25(-) (DN1), CD44(hi)CD25(+) (DN2), CD44(lo)CD25(+) (DN3) and CD44(lo)CD25(-) (DN4). Although it is well established that the TCR beta, gamma and delta genes are rearranged and expressed in association with the CD3 components in DN thymocytes, the precise timing of expression of the TCR and CD3 proteins has not been determined. In this report we have utilized a sensitive intracellular (ic) staining technique to analyze the expression of ic CD3epsilon, TCR beta and TCR gammadelta proteins in immature DN subsets. As expected from previous studies of TCR beta rearrangement and mRNA expression, icTCR beta(+) cells were first detected in the DN3 subset and their proportion increased thereafter. Surprisingly, however, both icCD3epsilon(+) and icTCR gammadelta(+) cells were detected at later stages of development than was predicted by molecular studies. In particular icCD3epsilon protein expression coincided with the transition from the DN2 to DN3 stage of development, whereas icTCR gammadelta protein expression was only detected in a minor subset of DN4 cells. The implications of these findings for alphabeta lineage divergence will be discussed.

Transcriptional regulation of CD4 gene expression by T cell factor-1/beta-catenin pathway.

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.

Successful vaccination against Boophilus microplus and Babesia bovis using recombinat antigens

Current methods for the control of the cattle tick Boophils microplus and the agent of bovine babesiosis, Babesia bovis are unsatisfactory. Effective immunological control of both parasites would have great advantages. However, naturally acquired immunity to the tick is generally unable to prevent serious production losses. A vaccine against the tick, based on a novel form of immunization, is being developed. A protective antigen has been isolated from the tick, characterized and produced as an effective, recombinant protein. A vaccine incorporating this antigen is currently undergoing field trials. In the Australian situation, improved tick control will probably increase endemic instability with respect to B. bovis. Fortunately, a trivalent, recombinant B. bovis vaccine has also been developed. This too is now undergoing pre-registration field trials.

Protection of aouts monkeys after immunization with recombinant antigens of Plasmodium falciparum

The genus Aotus spp. (owl monkey) is one of the WHO recommended experimental models for Plasmodium falciparum blood stage infection, especially relevant for vaccination studies with asexual blood stage antigens of this parasite. For several immunization trials with purified recombinant merozoite/schizont antigens, the susceptible Aouts kenotypes II, III, IV and VI were immunized with Escherichia coli derived fusion proteins containg partial sequences of the proteins MSAI (merozoite surface antigen I), SERP (serine-strech protein) and HRPII (histidine alanine rich protein II) as well as with a group of recombinant antigens obtained by an antiserum raised against a protective 41 kD protein band. The subcutaneous application (3x) of the antigen preparations was carried out in intact animals followed by splenectomy prior to challange, in order to increase the susceptibility of the experimental hosts to the parasite. A partial sequence of HRPII, the combination of three different fusion proteins of the 41 kD group and mixture of two sequences of SERP in the presence of the modified Al(OH)3 adjuvant conferred significant protection against a challange infection with P. falciparum blood stages (2-5 x 10 (elevado a sexta potência) i. RBC). Monkey immunized with the MS2-fusion protein carrying the N-terminal part of the 195 kD precursor of the major merozoite surface antigens induced only marginal protection showing some correlation between antibody titer and degree of parasitaemia. Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E. coli containing selected partial sequences of SERP, HRPII and MSAI. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding schizont polypeptides. In two independent immunization trials using 13 animals (age 7 months to 3 years) we could show that immunization of Aotus monkeys with either of the two hybrid proteins administered in an oil-based well tolerated formulation protected the animals frm a severe experimental P. falciparum (strain Palo Alto) infection.

Tegumental proteins of Schistosoma mansoni: complex biomolecules and potent antigens

The passive transfer of monoclonal antibodies, direct vaccination and in vitro assays have all shown that antigens associated with the tegumental membranes of Schistosoma mansoni are capable of mediating protective immune responses against the parasite in animal models. Furthermore, the principal antigens are highly antigenic during natural infection in man and stimulate strong humoral and cellular responses although, at present, their role in mediating protective immune responses in man remains equivocal. This presentation will review the current state of knowledge of the structure and expression of the major antigenic tegumental proteins of the schistosome and will attempt to relate the relevance of their structural features to possible function both in terms of protective immunity and parasite's ability to survive within the definitive host. A focus will be recent advances that have been made in the identification of means of anchoring of the antigenic proteins to the tegumental membrane. In addition, the implications of the structural complexity of the tegumental proteins in terms of their possible utility in vaccination and diagnosis will be considered.

The beta1 and beta3 integrins promote T cell receptor-mediated cytotoxic T lymphocyte activation.

Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling.